Affinity Separations
Of the separation technologies available today, those based on affinity interactions are ever more popular, particularly at the laboratory scale. Affinity separation has become the preferred method for purifying proteins and other biomolecules from complex, biologically derived fluids. Affinity Chromatography and Biological Recognition, I. M. Chaiken, M. Wilchek and I. Parikh (eds.), Academic Press, New York, 1983; Hill, E. A. and M. D. Hirtenstein, "Affinity chromatography: its application to industrial scale processes", Advances in Biotechnological Processes, Alan R. Liss, Inc., New York, 1983). The key to the method's attractiveness is its unequaled degree of selectivity.
Affinity separations, as they are conventionally practiced, typically involve a number of sequential steps. First, a solution containing a component to be separated from the solution (a component of interest) is passed through a column containing a highly specific ligand which will reversibly bind the compound of interest immobilized on a support, usually high-surface-area beads or particles. As the fluid passes through the column in this loading step, the desired component binds selectively and reversibly to the immobilized ligand, while most impurities pass unhindered. Residual impurities are removed by flushing the column with an appropriate buffer solution in a subsequent washing step. The component, now purified but still bound to the immobilized ligand, is then recovered by passing an eluent solution through the column that has the effect of disrupting the ligand-to-ligate binding interaction. Generally, the pH, concentration of a salt, or some other chemical characteristic of this eluent solution is altered significantly from the corresponding values of the loading and wash solutions, and it is this change that is responsible for weakening the affinity interaction and thereby causing desorption and elution of the ligate molecule.
Many types of molecules can serve as ligands, including antibodies, antigens, enzyme inhibitors, isolated receptors, and more recently, cloned receptors. Bailon, P. et al., Bio/Technology 5:1195 (1987). In contrast, however, the choice of materials to support the ligand has been somewhat limited. Agarose gel beads (e.g., 50 to 150 microns diameter) have traditionally received the most attention as affinity ligand supports, particularly on the laboratory scale. Within recent years, cross-linked and accordingly more rigid versions of these and other polysaccharide-based gel beads have been developed and introduced, as have various microporous support particles based on synthetic polymer compositions. These polymeric support materials are now complemented by various inorganic materials. For example, porous silica packed in high-pressure columns is used to perform affinity separations in an HPLC-like process. Typical pore diameters in the silica support range from about 200 to about 1000 Angstroms, whereas silica particle diameters are generally in the range of about 5 to 25 microns.
Affinity Membranes
Affinity separation processes for the recovery and purification of proteins are conventionally carried out using sorbent beads or particles packed in columns, as discussed above. The adsorption process is carried out in a cyclical fashion comprising four steps:
1. Load: A solution of target component in a mixture is made to pass through a packed column; target component ("ligate") is recognized and captured by the immobilized sorbent ("ligand"), while most contaminants pass through. PA1 2. Wash: A wash solution is passed through the column to flush out contaminants present in the column void volume as well as to displace non-specifically bound contaminating substances. PA1 3. Elute: An eluent solution is passed through the column to disrupt the affinity binding between immobilized ligand and reversibly bound ligate, causing elution of the latter from the column in a purified condition. PA1 4. Regenerate: A regeneration solution is passed through the column in order to return it to conditions (e.g., pH and/or ionic strength) that favor ligand/ligate binding.
Despite the high selectivity that affinity processes provide, however, their application on the process scale has been hampered by the inability of affinity columns to handle high flowrates at reasonable ligand utilization efficiencies.
Affinity membrane devices are based on microporous membranes, preferably hollow fibers activated by covalent attachment of affinity ligands to the interior surfaces of the membrane's pore walls. In operation, feed solution is made to flow through the membrane from one of its surfaces to the other, during which process the target molecule is recognized and captured by the immobilized ligand which it encounters, leaving the filtrate devoid of ligate. Like columns, these affinity membranes can be operated in a cyclic affinity adsorption process to produce high-purity protein in a single step. However, unlike columns based on particulate affinity ligand supports, affinity membranes are not hampered by the serious pressure drop and mass transfer limitations from which columns suffer. As a result, affinity membranes are capable of operating at higher volumetric throughputs and ligand utilization efficiencies than are columns.
Both polymeric and inorganic affinity support particles suffer from hydrodynamic or pressure drop limitations when used in columns. With the former (e.g., soft agarose gel beads), particle compressibility is a problem, inasmuch as attempts to increase flowrate through a column packed with agarose are normally met by increased pressure drops. This leads in turn to further compression of the particles and reduced bed permeability. Clonis, Y.D., Bio/Technology 5:1290(1987). It is only by resorting to very shallow but large-diameter packed columns (i.e., columns with a relatively large ratio of bed diameter to depth) that practical volumetric throughputs can be obtained. Alternatively, one can resort to more rigid particles (e.g., silica, controlled-pore glass), but here the small size of the support particles limits volumetric throughput unless high operating pressure is employed.
In contrast, affinity membranes with adsorptive pore walls provide extremely short fluid-flow path lengths in comparison to the superficial area provided for flow. This unique geometry of affinity membranes thus leads to very high fluid throughputs per unit of applied pressure difference as compared to affinity columns.
Another important consideration in evaluating the merits of membranes vs. columns as affinity ligand supports is the matter of their relative mass transfer efficiency. Efficient capture of a target protein in an affinity column requires that the characteristic time for diffusion of protein to the immobilized ligand be short as compared to the residence time of fluid in the column. If this condition is not met, premature breakthrough is encountered and the "dynamic" sorption capacity of the bed will not approach its "static" or equilibrium capacity.
A characteristic diffusion time for the encounter between diffusing ligate and immobilized ligand can be defined as the ratio of the square of a characteristic diffusion distance to the diffusivity of the ligate molecule. The required residence time of fluid in the affinity device during the loading step will increase in proportion to this characteristic diffusion time. Thus, in order to keep the characteristic distance for ligate diffusion into the support as small as possible (and thereby to maximize device throughput during loading), it is necessary to use support particles that are as small as practical (e.g., fine silica or synthetic polymeric particles). However, doing so tends to aggravate the above-mentioned pressure drop problem, forcing one away from low-pressure operation towards a high-pressure liquid chromatography process.
In contrast, affinity membranes obviate the need to work with small (e.g., micron-sized) particles in order to minimize diffusion distances and diffusion times. Where protein-containing solutions are pumped across affinity membranes, the characteristic distance across which ligate must diffuse in order to meet membrane-bound ligand is of the order of a quarter of the pore diameter; typically, this diffusion distance is only a fraction of a micron. Because diffusion time varies with the square of diffusion distance, the impact of the reduction in diffusion distance afforded by affinity membranes on improved mass transfer efficiency and volumetric productivity is dramatic. These and other aspects of affinity membrane performance have been discussed by S. Brandt et al., Bio/Technology 6:152 (1988) and in co-pending U.S. applications Ser. No. 07/265,061 and No. 07/428,263, referred to above.